Butyrate-induced alterations of phosphoinositide metabolism, protein kinase C activity and reduced CD44 variant expression in HT-29 colon cancer cells

Abstract

Abstract. Initiation of cell growth and neoplastic transformation frequently involves activation of growth factor receptor-coupled tyrosine kinases and stimulation of the phosphoinositide second messenger system. Altered expression of CD44 variants was reported in several malignant tumor types with possible implications for tumor progression and prognosis. CD44 variant expression was reported to be associated with second messenger activation and differentiation. We therefore investigated the effects of butyrateinduced short-term differentiation on phosphoinositide signaling, phospholipase C and protein kinase C activity and alteration of CD44 variant expression in human HT-29 colon carcinoma cells. HT-29 cells were cultured with sodium butyrate for 6 days. Phosphoinositide turnover was measured by [32 P]orthophosphate incorporation and phospholipase C activity by determination of the release of [ 3 H]inositolphosphates from [ 3 H]myoinositol prelabeled cells. Protein kinase C activity was determined by histone III-S phosphorylation, PKC subtype expression by RNase protection analysis, and CD44 variant expression was determined by RT-PCR using variant-specific primers. Treatment of HT-29 human colon carcinoma cells with sodium butyrate caused a dose-dependent inhibition of cell proliferation (IC 50 , 2.5 mM) with morphologic signs of an enterocytic differentiation following 6 days of treatment. The phosphoinositide turnover as determined by 32 P-incorporation under non-equilibrium conditions showed a 30-40% inhibition of labeled phosphoinositides and phosphatidic acid and a dose-dependent inhibition of cholinergically stimulated phospholipase C activity as a secondary event following butyrate-induced enterocytic differentiation. However, long-term incubation of HT-29 cells with phorbol ester or an inhibitor of classical and novel PKC subtypes did not affect cell proliferation. In butyrate-treated HT-29 cells activation of calcium-dependent protein kinase C by cholinergic stimulation or phorbolester treatment induced an increase in membrane-bound cPKC activity, while expression of distinct high-molecular CD44 variant transcripts v3 (670 bp), v5 (940 bp) and v8 (535 bp) were drastically reduced after butyrate pretreatment. Enterocytic differentiation of HT-29 colon carcinoma cells seems to be associated with alterations in phosphoinositide resynthesis, phospholipase C activity and ligand/receptorinduced PKC translocation. The observed reduction of distinct high-molecular CD44v3, v5 and v8 variants following butyrate-induced differentiation indicates an association of specific CD44 variant expression with the malignant phenotype of HT-29 colon cancer cells, thus being possible targets for new diagnostic and therapeutic strategies.

Bean (Phaseolus vulgaris L.) polysaccharides modulate gene expression in human colon cancer cells (HT-29)

“…MSH2 gene was up-regulated (9.8-fold) by FE-hgf-LC 50 extract supporting its involvement with DNA repair in accordance with earlier observations (Hoeijmakers, 2001;Lyer, Pluciennik, Burdett, & Modrich, 2006;Peltomäki, 2001Peltomäki, , 2003. Several studies (Emenaker et al, 2001;Hinnebusch, Meng, Wu, Archer, & Hodin, 2002;Hofmanová, Vaculová, Lojek, & Kozubík, 2005;Koop, Fichter, Assert, Pfeiffer, & Classen, 2009;Li, Luo, Paul, Tang, & Yuan, 2006) suggest that the production of butyrate in the colon may be protective against colon carcinogenesis. Current studies and clinical trials (Archer et al, 2005;Butler et al, 2001;Cucciolla et al, 2008;He et al, 2001;Law, Lai, Lui, Wan, & Wong, 2008;Marks, Rifkind, Richon, & Breslow, 2001) strongly suggest that histone deacetylace inhibitors (HDAC) such as trichostatine (TSA), butyrate and suberoylanilide hydroxamic acid, which also induce p21Waf1/Cip1 expression, are effective in arresting cancer cell proliferation and lead to cells undergoing differentiation (as in acute promielocytic anemia) or apoptosis.…”

Frequent expression of the high molecular, 673-bp CD44v3,v8-10 variant in colorectal adenomas and carcinomas

“…We previously showed, that the expression of CD44 variant isoforms is stimulated by growth factors like PDGF or IGF-I and regulated by phospho-inositide 3-kinase and protein kinase C in SK-N-SH human neuroblastoma cells (18). Recently, we reported, that sodium butyrate, a potent HDAC inhibitor, known to regulate intestinal epithelial cell proliferation and differentiation, reduced the expression of high molecular CD44v3, v5 and v8 variants in HT-29 colon carcinoma cells (29). Similar findings were reported by Barishat et al in HM7 colon carcinoma cells showing decreased expression of CD44 variants and reduced metastatic potential following butyrate treatment, indicating an association of CD44 variant overexpression with hyperproliferative and undifferentiated cells (30).…”

Associations between markers of colorectal cancer stem cells, mutation, microRNA and the clinical features of ulcerative colitis

“…This area of research has implicated cancer stem cells (CSCs), microRNA (miRNA) and genetic mutations as markers of the development of CRC. Studies have identified CD44 as a specific marker for CSCs and have also found several CD44 splice variants, some of which correlated well with colon cancer progression [11][12][13]. Our previous study reported increased expression of CD166 with CD44 to be associated with colonic carcinogenesis.…”